The purpose of these studies is to develop imaging techniques to monitor sub-cellular structures and processes, in vivo. We have been systematically developing an in vivo optical microscopy system that is adapted to biological tissues and structures rather than forcing an animal on a conventional microscope stage. The following major findings were made over the last year: 1) The tight coupling of mitochondria across muscle cells in the mitochondria reticulum is a risk to the cell. If one mitochondria fails, it could pull down the entire mitochondrial network just like a short circuit in a house. We have recently completed a study that demonstrates a rapid fail safe system is in place that removed damaged mitochondria from the network. Our current hypothesis is that this fail safe, or circuit breaker, mechanism is structural in nature representing the physical uncoupling of the mitochondria from the network and cytoskeleton. This is based on the observation that the mitochondrial reticulum is under tension, that is stretched by the cytoskeleton to hold these complex positions, until damage is detected and the mitochondria is released and springs back to its native spherical structure. This process has been directly observed in muscle cells during local disruption of mitochondrial function. This adds an entirely new regulatory aspect to the mitochondria dealing with it distribution and structure to meet cellular needs. We are currently modifying a STED microscope to conduct these studies at super resolution optical microscopy (25 nm) to further characterize this cellular process. 2) Using our ability to monitor subcellular events rapidly in the living animal, we have completed a collaboration with Dr. Sinnis at Johns Hopkins to monitor the trafficking of malaria parasites upon inject via a simulated mosquito proboscis. The simulated proboscis is a specially designed fluorescent glass pipet that we can monitor in the animal with a 2-Photon excitation microscope.3) We are modifying our multiphoton system originally used for water CARS to perform stimulated fluorescence and Raman spectroscopy on intact cellular systems. This will permit adequate signal to noise to hopefully measure the subcellular distribution of chromophores detected in absorbance or Raman spectroscopy greatly improving our understanding of the compartmentalization of cellular metabolic process.